Following sEV preparation by PEG precipitation and ultracentrifugation from the supernatant of hCMEC/D3, as described, sEV preparations were labeled with PKH67 membrane dye (Sigma-Aldrich). Briefly, sEV preparations were stained with 2 µM PKH67 diluted in diluent C for 5 min at room temperature. Excess dye was removed by washing twice with PBS using Amicon-Ultra Centrifugal Filter Units (Merck Millipore). Labeled sEVs were incubated with hCMEC/D3 for 24 h. Cells were fixed with PFA, labeled with rabbit anti-Rab7 (D95F2; Cell Signaling Technology), rabbit anti-CD63 (LS-C204227; Lifespan Biosciences) or rabbit anti-LAMP1 (1D4B; Abcam) antibody, detected by appropriate secondary antibody (as above) and counterstained with Hoechst 33342 (Thermo Fisher Scientific).
Rabbit anti rab7 d95f2
Manufactured by Cell Signaling Technology
Rabbit anti-Rab7 (D95F2) is a primary antibody that recognizes the Rab7 protein. Rab7 is a small GTPase that regulates late endocytic/lysosomal trafficking. The antibody was generated by immunizing rabbits with a synthetic peptide corresponding to residues surrounding Asp95 of human Rab7.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (2 mentions)
Pkh67 membrane dye, by Merck Group (2 mentions)
Rabbit anti cd63 ls c204227, by LifeSpan BioSciences (2 mentions)
Rabbit anti lamp1 1d4b, by Abcam (2 mentions)
Rabbit anti eea1 c45b10, by Cell Signaling Technology (1 mentions)
2 protocols using rabbit anti rab7 d95f2
1
Labeling and Imaging of Extracellular Vesicles
2
Tracking sEV Uptake in hCMEC/D3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Following sEV preparation by PEG precipitation and ultracentrifugation from the supernatant of hCMEC/D3, as described, sEV preparations were labeled with PKH67 membrane dye (Sigma-Aldrich). Briefly, sEV preparations were stained with 2 µM PKH67 diluted in diluent C for 5 min at room temperature. Excess dye was removed by washing twice with PBS using Amicon-Ultra Centrifugal Filter Units (Merck Millipore). Labeled sEVs were incubated with hCMEC/D3 for 24 hours. Cells were fixed with PFA, labeled with rabbit anti-EEA1 (C45B10; Cell Signaling Technology), rabbit anti-Rab7 (D95F2; Cell Signaling Technology), rabbit anti-CD63 (LS-C204227; Lifespan Biosciences) or rabbit anti-Lamp1 (1D4B; Abcam) antibody, detected by appropriate secondary antibody (as above) and counterstained with Hoechst 33342 (Thermo Fisher Scientific).
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